Probiotic bacteria

ABSTRACT

Strains of pro-biotic bacteria, their isolation, characteristics and methods of use to prevent or treat carriage by a food production animal of  Salmonella  that causes human salmonellosis are provided. Methods for isolating and characterizing the probiotic bacteria are also provided. The present invention further provides methods for using the probiotic bacteria to prevent or treat  Salmonella  strains that cause human salmonellosis found in food production animals.

FIELD OF THE INVENTION

This invention relates generally to the control of pathogenic bacteriain animals raised for human consumption and more particularly to thecontrol of Salmonella enterica serovar Typhimurium DT104 and Salmonellaenterica serovar Newport by administering probiotic bacteria to theanimals.

BACKGROUND OF THE INVENTION

Salmonella spp. are widespread with in the environment. Their primaryhabitat is the intestinal tracts of birds, reptiles, animals (especiallythose on the farm), humans, and occasionally insects. They may also befound in other parts of the body from time to time, for example in thespleen, liver, bile, mesenteric and portal lymph nodes, diaphragm, andpillar in slaughterhouse pigs. Jay, J. M., Modern Food Microbiology, 6thed. Aspen Publishers, Gaithersburg, Md. (2000).

Serovars that cause human salmonellosis are most often found in foods ofanimal origin, such as pork and poultry meats, and dairy products.Oosterom, J., Int. J. Food Microbiol. 12:41-52 (1991). The persistenceof salmonellae in slaughterhouses and meat processing facilitiescontinues due to the exposure of livestock to environmental sources ofcontamination, contaminated feeds, and parental transmission ofinfection. The feces of infected humans and animals contaminate watersources, which subsequently infect farm animals, then contaminate meatduring slaughter, and subsequently infect humans, beginning the cycleanew. This cycle is augmented by the practice of international shippingof animal products and feed, which has lead to the worldwidedistribution of salmonellosis.

In the 1980s, surveillance data of cattle and human isolates indicatethat Salmonella enterica serovar Typhimurium DT104 emerged worldwide. S.Typhimurium DT104 typically is resistant to the antibiotics ampicillin,chloramphenicol, streptomycin, sulphonamides and teracycline (R-typeACSSuT). Threlfall, E. J. et al., Vet. Rec. 134:577 (1994). Currently,data suggest that a multi-resistant Salmonella enterica serovar Newportis emerging in the United States. S. Newport typically is resistant toat least nine antibiotics. Recent studies revealed that 3.5% of retailground beef was positive for Salmonella spp. of which 35.6% was S.Typhimurium DT104. Zhao T. et al., J. Food Prot. 65:403-407 (2002).Between January and April 2002, a five-state outbreak of S. Newportoccurred. Exposure to raw or undercooked ground beef was implicated asthe vehicle of transmission. Cattle are thought to be a primaryreservoir through which both these multi-resistant pathogens can enterthe food supply.

Clinical symptoms of S. Typhimurium DT104 in humans include diarrhea,fever headache, nausea, vomiting and abdominal pain. One-fourth ofpatients infected in a case-control study had bloody diarrhea, 41% ofpatients required hospitalization, and 3% of patients died. This is muchhigher than the case-fatality rate associated for non-typhoid Salmonellainfections, which other than for DT104, is approximately 0.1%. Aldina,J. E. et al., J. Am. Vet. Med. Assoc. 214:790-798 (1999).

Surveys of feedlot cattle in the United States done in 1998 revealedthat 38% of feedlots were Salmonella spp. positive, and 5.5% of allfecal samples collected were positive for Salmonella spp. S. TyphimuriumDT104 was detected in 2.6% of the feedlots, and 2.9% of the positivefecal samples. Fedorka-Cray, P. J. et al., J. Food Prot. 61:525-530(1998). A similar study conducted on beef cattle in 2000 revealed that11.2% of all operations tested positive for Salmonella spp., and 1.4% ofall fecal samples were positive. Dargatz, D. A. et al., J. Food Prot.63:1648-1653 (2000). There are clear associations between S. TyphimuriumDT104 infection in food production animals and humans. Davis, A. et al.,Communicable Disease Report CDR Rev. 6:159-162 (1996).

SUMMARY OF THE INVENTION

Strains of probiotic bacteria, their isolation, characteristics andmethods of use to prevent or treat carriage by a food production animalof Salmonella that causes human salmonellosis are provided. Anon-limiting example of Salmonella strains that cause humansalmonellosis are Salmonella enterica Typhimurium DT104 or Newport. By“probiotic” it is meant bacteria having the property of preventingestablishment of Salmonella in a food production animal previouslyadministered an effective dose of said probiotic bacteria. Strains ofprobiotic bacteria that inhibit the growth of Salmonella strains thatcause human salmonellosis can be strains of E. coli and Bacilluscirculans.

The present invention also provides a method for preventing the carriageby a food production animal of Salmonella strains that cause humansalmonellosis. The method comprises the step of administering aneffective amount of a strain or combination of strains of probioticbacteria to the food production animal prior to exposure to Salmonellastrains that cause human salmonellosis.

The invention further provides a method for reducing or eliminating froma food source animal Salmonella strains that cause human salmonellosisby administering an effective amount of a strain or combination ofstrains of probiotic bacteria. The method is useful to maintain herds orflocks of animals free of Salmonella strains that cause humansalmonellosis and reduce carriage and fecal shedding of Salmonellastrains that cause human salmonellosis prior to slaughter.

The administration of probiotic bacteria is accomplished by feeding afeed supplement or additive which comprises an effective amount ofprobiotic bacteria, or by supplying a water treatment additive orinoculum to the animals' drinking water. The invention thereforeprovides a feed supplement composition comprising probiotic bacteria anda water additive comprising probiotic bacteria.

Additional objects, advantages, and features of the present inventionwill become apparent from the following description and appended claims,taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent toone skilled in the art by reading the following specification andsubjoined claims and by referencing the following drawings in which:

FIG. 1A is a photograph of a paper disk assay showing the zones ofinhibition of S. Typhimurium DT104 by isolate 36-1 on TSA agar;

FIG. 1B is a photograph of a paper disk assay showing the zones ofinhibition of S. Typhimurium DT104 by isolate 36-1 on XLD agar;

FIG. 2A is a photograph of a agar-spot assay showing the zones ofinhibition of S. Typhimurium DT104 by isolate 59-9 on TSA agar;

FIG. 2B is a photograph of a agar-spot assay showing the zones ofinhibition of S. Typhimurium DT104 by isolate 59-9 on MAC agar;

FIG. 3 shows the an agarose gel of the comparison of PFGE DNA pulsotypeof gram-negative competitive inhibition isolates from cattle;

FIG. 4A is a graph showing the growth of Salmonella sp. at 37° C. inbovine feces with a low inoculum of DT104 and probiotic bacteria;

FIG. 4B is a graph showing the growth of Salmonella sp. at 37° C. inbovine feces with a high inoculum of DT104 and probiotic bacteria;

FIG. 4C is a graph showing the growth of Salmonella sp. at 37° C. inbovine feces with S. Newport and probiotic bacteria;

FIG. 5A is a graph showing the growth of Salmonella sp. at 21° C. inbovine feces with a low inoculum of DT104 and probiotic bacteria;

FIG. 5B is a graph showing the growth of Salmonella sp. at 21° C. inbovine feces with a high inoculum of DT104 and probiotic bacteria; and

FIG. 5C is a graph showing the growth of Salmonella sp. at 21° C. inbovine feces with S. Newport and probiotic bacteria;.

DETAILED DESCRIPTION OF THE INVENTION

Strains of probiotic bacteria, their isolation, characteristics andmethods of use to prevent or treat carriage by a food production animalof Salmonella that causes human salmonellosis are provided. Theprobiotic bacteria and methods of the present invention are especiallyeffective for preventing and or treating carriage of Salmonella strainsthat cause human salmonellosis and have multiple antibiotic resistence.A non-limiting example of Salmonella strains that cause humansalmonellosis are Salmonella enterica Typhimurium DT104 or Newport.

“Food production animal” is used herein to describe any mammal or avianthat is prepared and used for human consumption. A food productionanimal can be, but not limited to, a ruminant animal such as beef anddairy cattle, pigs, lamb, chicken, turkey or any other fowl.

“Probiotic” is used herein as an adjective to describe bacteria isolatedfrom a natural source and having the property of inhibiting the growthof Salmonella strains that cause human salmonellosis. The test of aninhibition used herein was an in vitro test on solid medium in whichculture supernatants of candidate isolated bacteria were observed fortheir property of inhibiting Salmonella enterica Typhimurium DT104 orNewport growth when applied to the surface of the solid medium.Typically, a paper disc impregnated with the culture supernatant of acandidate strain was placed on the surface of an agar plate seeded witheither Salmonella enterica Typhimurium DT104 or Newport. Probioticbacterial supernatants caused a ring of clear agar or of reduced growthdensity indicating inhibition of Salmonella enterica Typhimurium DT104or Newport in the vicinity of the disc. There are other tests forinhibition which are available or could be devised, including directgrowth competition tests, in vitro or in vivo which can generate a panelof probiotic bacteria similar to that described herein. The bacterialstrains identified by any such test are within the category of probioticbacteria, as the term is used herein.

The term “dominant probiotic” is applied to probiotic bacteria whichpersist in, and are re-isolatable from an animal to which the bacteriahave been administered. For example, bovine calves can be fed a mixtureof probiotic strains, then from a variety of tissues, digestive contentsand feces are sampled 26 days post-inoculation. Recovered strains aredesignated dominant probiotic strains. Other criteria can be employed,including shorter or longer time periods between inoculation andsampling. It is advisable to choose a time period sufficiently long thatpersistence of dominant probiotic strains can provide useful reductionof the amount of Salmonella strains that cause human salmonellosiscarried by the animal.

Isolation of probiotic bacteria can be carried out by those of ordinaryskill in the art, following the principles and procedures describedherein. Of 1097 colonies isolated from cattle feces and tissues, sixgram-positive isolates and 24 gram-negative isolates were identified asprobiotic bacteria. Eight of the isolates, 31-6, 47-10, 50-10, 58-9,59-9 small, 59-9 big, 71-8 and 76-9 were better at inhibiting Salmonellaenterica Typhimurium DT104 or Newport. Therefore, the testing of similarnumbers of independent isolates is reasonably likely to successfullyyield probiotic bacteria. Probiotic bacteria isolates 31-6, 76-9 and58-9 have been deposited with the American Type Culture Collection(ATCC), 1080 University Boulevard, Manassas, Va. 20110-2209, under theterms of the Budapest Treaty, and has been accorded the ATCC designationnumbers , ,and , respectively. The deposit will be maintained in theATCC depository, which is a public depository, for a period of 30 years,or 5 years after the most recent request, or for the effective life of apatent, whichever is longer, and will be replaced if the deposit becomesdepleted or nonviable during that period. Samples of the deposit willbecome available to the public and all restrictions imposed on access tothe deposit will be removed upon grant of a patent on this application.

The probiotic bacteria can be any type of bacteria and may notnecessarily be a different strain of Salmonella. For example, of theprobiotic bacteria described herein, 22 of the isolates were identifiedas E. coli, and 7 were identified as Bacillus circulans. Administrationof probiotic bacteria can be accomplished by any method likely tointroduce the organisms into the digestive tract. The bacteria can bemixed with a carrier and applied to liquid or solid feed or to drinkingwater. The carrier material should be non-toxic to the bacteria and theanimal. Preferably, the carrier contains an ingredient that promotesviability of the bacteria during storage. The bacteria can also beformulated as an inoculant paste to be directly injected into ananimal's mouth. The formulation can include added ingredients to improvepalatability, improve shelf-life, impart nutritional benefits, and thelike. If a reproducible and measured dose is desired, the bacteria canbe administered by a rumen cannula. The amount of probiotic bacteria tobe administered is governed by factors affecting efficacy. Whenadministered in feed or drinking water the dosage can be spread over aperiod of days or even weeks. The cumulative effect of lower dosesadministered over several days can be greater than a single larger dosethereof. By monitoring the numbers of Salmonella strains that causehuman salmonellosis in feces before, during and after administration ofdominant probiotic bacteria, those skilled in the art can readilyascertain the dosage level needed to reduce the amount of Salmonellastrains that cause human salmonellosis carried by the animals. One ormore strains of dominant probiotic bacteria can be administeredtogether. A combination of strains can be advantageous becauseindividual animals may differ as to the strain which is most persistentin a given individual.

Probiotic bacteria can be administered as a preventive, to preventanimals not presently carrying Salmonella strains that cause humansalmonellosis from acquiring the strains by exposure to other animals orenvironments where the strains are present. Young and mature foodproduction animals about to be transferred to a new location, such as afeed lot, are attractive candidates for preventive administration.

Treatment of animals carrying Salmonella strains that cause humansalmonellosis can be accomplished to reduce or eliminate the amount ofthe strains carried by the animals, by administering probiotic bacteriato animals infected with Salmonella strains that cause humansalmonellosis. Animals known to be shedding the strains in feces, orthose raised where the strains are known to exist are suitablecandidates for treatment with probiotic bacteria.

The methods for administering probiotic bacteria are essentially thesame, whether for prevention or treatment. Therefore, the need to firstdetermine whether the undesired Salmonella strains are being carried bythe animals is removed. By routinely administering an effective dose toall the animals of a herd, the risk of contamination by the undesiredSalmonella strains can be substantially reduced or eliminated by acombination of prevention and treatment.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following examples, which arepresented for purposes of illustration and not by way of limitation.

EXAMPLE 1 Isolation and Identification of Salmonella spp. From BovineFeces

Methods:

Sample collection. A total of 108 fecal samples were collected in themiddle Georgia region from September 2001 though January 2002. Sampleswere obtained from 28 dairy cattle, 80 beef cattle and five calvesbetween four months and one year of age. Ten grams of feces wascollected into Cary Blair with indicator fecal transport system(Corpimex, Miami, Fla.), and immediately transported at 5° C. Sampleswere stored at 4° C. for 0 to 7 days until use.

Salmonella isolation and identification. Each fecal sample (10 g) waspreenriched in 90 ml of lactose broth (Becton Dickinson, Sparks, Md.)for 24 hours at 35° C. After preenrichment, 1-ml volumes of enrichmentculture were transferred, for selective enrichment, to 10 ml of selenitecystine broth (Becton Dickinson, Sparks, Md.) and incubated for 24 hoursat 37° C., to 10 ml of tetrathionate broth (Becton Dickinson, Sparks,Md.) and incubated for 48 hours at 37° C., and to 10 ml ofRappaport-Vassiliadis R10 broth (Becton Dickinson, Sparks, Md.) andincubated for 24 hours at 42° C. After selective enrichment, a 10-μlloopful from each broth was plated in duplicate on to the surface ofbismuth sulfite agar (BSA), Hektoen enteric agar (HEA), xylose lysinedeoxycholate agar (XLD) and xylose lysine tergitol 4 agar (XLT4) (allBecton Dickinson, Sparks, Md.) plates. Plates were incubated for 24hours at 37° C. Colonies with typical Salmonella spp. morphology wereselected from all plates, no more than 10 colonies per plate, andtransferred into triple sugar iron agar and lysine iron agar (bothBecton Dickinson, Sparks, Md.) slants and incubated for 24 hours at 35°C. All presumptive Salmonella isolates were tested by the Salmonellalatex agglutination assay (Oxoid Ltd., Basingstoke, Hampshire, UK). Allisolates positive with the Salmonella latex agglutination assay weretested with the API 20E assay (bioMerieux, Hazelwood, Mo.) forbiochemical characteristics for the identification of Salmonella. Zhao,T. et al., J. Food Prot. 65:403-407 (2002). Serotyping was conducted atthe U.S. Department of Agriculture-Animal and Plant Health InspectionService (APHIS) National Veterinary Services Laboratories, Ames, Iowa.Antibiotic resistance profiles were conducted at the U.S. Department ofAgriculture-Agricultural Research Service, Athens, Ga.

Results

Salmonella spp. were isolated from 10 of 108 fecal samples. All positivesamples were from beef cattle over one year of age (Table 1), and werecollected from an auction market. Two samples were collected on Oct. 16,2001, three samples were collected Jan. 15, 2002, and five samples werecollected Jan. 29, 2002. TABLE 1 Serotype, serogroup and antibioticresistance of Salmonella spp. isolates Isolate Antibiotic No. Dateisolated Serotype Serogroup resistance^(ab) 55 Oct. 16, 2001 Newport C2AAmCeCfCpC SSuT 57 Oct. 16, 2001 Newport C2 AAmCeCfCpC SSuT 73 Jan. 15,2002 Bareilly C1 None 74 Jan. 15, 2002 Mbandaka C1 None 78 Jan. 15, 2002Newport C2 AAmCeCfCpC STTr 88 Jan. 29, 2002 Newport C2 AAmCeCfCpC SSuTTr90 Jan. 29, 2002 Montevideo C1 None 92 Jan. 29, 2002 Meleagridis E None102 Jan. 29, 2002 Monophasic B None 103 Jan. 29, 2002 Monophasic B None^(a)A = ampicillin, Am = amoxicillin/clavulanic acid, Ce = cefoxitin,Cf-ceftiofur, Cp = cephalothin, C = chloramphenicol, S = streptomycin,Su = sulphamethoxazole, T = tetracycline Tr =trimethoprim/sulphamethoxzole^(b)Screened against, amikacin, amoxicillin/clavulanic acid, ampicillin,apramycin, cefoxitin, ceftiofur, ceftriazone, cephalothin,chloramphenicol, ciprofloxacin, gentamicin, inipenem, kanamycin,nalidixic acid, streptomycin, sulphamethoxazole, tetracycline,trimethoprim/sulphamethoxazole

The positive isolates included serogroups B, C1, C2, and E, four ofwhich were serotyped as Salmonella Newport, two as monophasic Salmonellasp., one as Salmonella Bareilly, one as Salmonella Mbandaka, one asSalmonella Montevideo, and one as Salmonella Meleaglidis.

Antimicrobial resistance profiles indicated that all four of theSalmonella Newport isolates were resistant to amoxicillin/clavulanicacid, ampicillin, cefoxitin, ceftiofur, cephalothin, chloramphenicol,streptomycin, and tetracycline. Isolates S55 and S57 were additionallyresistant to sulphamethoxazole, and isolate S78 was also resistant totrimethoprim/ sulphamethoxazole. Isolate S88 was additionally resistantto sulphamethoxazole and trimethoprim/sulphamethoxazole, and hadintermediate resistance to ceftriazone. All other Salmonella isolateshad intermediate resistance to tetracycline, but were sensitive to allother antibiotics. All isolates were sensitive to amikaxin, apramycin,ciprofloxacin, gentamicin, imipenem, kanamycin, and naladixic acid.

EXAMPLE 2 Isolation and Identification of Competitive InhibitionBacteria

Methods

Isolation of potential competitive inhibition bacteria.Salmonella-negative fecal samples were serially diluted (1:10) in 0.1%peptone buffer, 0.1 ml of each dilution was plated in duplicate ontoMacConkey agar (MAC) and tryptic soy agar (TSA) (both Becton Dickinson,Sparks, Md.), and the plates were incubated for 24 hours at 37° C. Sevencolonies were randomly selected from MAC agar plates, and three colonieswere randomly selected from TSA plates. Each colony was transferred to atest tube containing 10 ml of trypic soy broth (TSB) (Becton Dickinson,Sparks, Md.) and incubated for 24 hours at 37° C.

Screening of cultures for anti-Salmonella Typhimurium DT104 properties.A three-strain mixture of Salmonella enteritidis serovar TyphimuriumDT104 from our culture collection, including strains 8748A-1 (cattleisolate, R-type ACSSuT), 11942A-1 (cattle isolate, R-type ACSSuT), and62 (ground beef isolate, R-type ASSuT), were initially used to screencultures for anti-Salmonella Typhimurium DT104 activity. Two methodswere used to screen for activity, the disk method (Zhao, T. et al., J.Clin. Microbiol. 36:641-647 (1998)) and the agar spot test (Schillinger,U. et al.,

Appl. Environ. Microbiol. 55:1901-1906 (1989)).

Approximately 10⁷ S. Typhimurium DT104 cells of approximately equalpopulations of each strain in 0.1 ml were plated onto the surfaces ofXLD and TSA plates and allowed to dry for at least 30 minutes.Supernatant fluid from each culture was filter sterilized(0.2-μl-pore-size cellulose acetate membrane: Nalgene Co., RochesterN.Y.) for determination of anti-S. Typhimurium DT104 activity. Two disks(12 mm diameter, Dispens-O-Disc, Difco Laboratories, Detroit, Mich.)were placed on the surface of both the TSA and XLD plates, and 0.1 ml ofthe filter-sterilized supernatant fluid from a single culture wasapplied to the surface of the disk. In addition, filter-sterilizedsupernatant fluid from E. coli ATCC 14763 (produces colicin V) and 70%ethanol were used as positive controls, and filter-sterilized TSB wasused as the negative control. Cultures were incubated for 24 hours at37° C. and observed for zones of growth inhibition. Competitiveinhibition bacteria were selected as those that produced a clear zone ofat least 1 mm around the disk.

Isolates were streaked onto TSA for single colonies and incubated for 24hours at 37° C. Single colonies were spot inoculated onto TSA and MACplates and incubated for 24 hours at 37° C. for colony development. Fivemilliliters of brain heart infusion broth (BHI) (Becton Dickinson,Sparks, Md.) with 0.5% agar (Becton Dickinson, Sparks, Md.) containingapproximately 106 CFU of the three strain S. Typhimurium DT104 mixtureat 50° C. was applied onto the surface of each plate, without disturbingthe colony, and allowed to cool. Plates were incubated for 24 hours at37° C., and observed for zones of growth inhibition. Competitiveinhibition bacteria were selected as those that produced a clear zone ofat least 1 mm around the disk.

Competitive inhibition cultures were then screened, using the methodsdescribed above, against nine additional strains of S. TyphimuriumDT104, obtained from the collection of P.J. Fedorka-Cray, U.S.Department of Agriculture-Agricultural Research Service, Athens, Ga. Allstrains were cattle isolates, and included from 1998 strains 526-K,2848-K and 12993-K, from 1999 strains MH25382, 99-103712-5 and 12-410and from 2000 strains 4698-K, NE14055, IA45025. Competitive inhibitioncultures were screened against 10 Salmonella spp. isolates obtainedduring the screening process of this study.

The pH of TSB was determined before and after culture growth. The pH ofTSB and MAC plates was determined before colony growth, and after colonygrowth, both near the colony and 2 cm away from the colony following 24hours of growth at 37° C. Growth curves for the competitive inhibitionisolates strains and Salmonella were performed and generation times werecalculated.

Identification of competitive inhibition bacteria. Initially competitiveinhibition isolates were characterized by Gram staining. Gram-positivestrains were subjected to catalase tests, oxidase tests, and tobiochemical testing using the API 50CH Assay (bioMerieux, Hazelwood,Mo.) with both the CHL media for lactic acid bacteria, and the CHB/Emedia for Bacillus spp. Spore formation was determined by holdingovernight cultures at 80° C. with agitation at 190 rpm for 10 minutes,then streaking the cultures in duplicate onto TSA plates and incubatingfor 24 hours at 37° C.

Gram-negative isolates were subjected to biochemical testing using theAPI 20E Assay (bioMerieux, Hazelwood, Mo.), and subtyping by PFGE usingprocedures similar to those described previously (Meng, J. et al., J.Med. Microbiol. 42:258-263 (1995)) and those used by the Centers forDisease Control and Prevention. This involved growing isolates on TSAplates for 24 hours at 37° C., then suspending cells of each culture inCell Suspension Buffer (CSB) (100 mM Tris: 100 MM EDTA, pH 8.0) with asterile swab to a cell populations having an optical density of 1.3-1.4at 610 nm (SPEC). The bacterial suspension, 0.2 ml, was mixed with 10 μlof 20 mg proteinase K/ml and 0.2 ml 1% SeaKem Gold: 1% SDS agarose in TEbuffer (1 mM Tris:1 mM EDTA, pH 8.0). The mixture was dispensed intosample moulds and the agarose plugs were digested with 0.1 mg proteinaseK/ml in lysis buffer (20 mM Tris:50 mM EDTA, pH 8.0+1% Sarcosine at 54°C. for 2 hours. The plugs were then washed at 50° C., three times insterile water and three times in TE buffer. Plugs were cut to 2.5 mmwide, prerestricted with IX restriction buffer for 10 minutes at 37° C.,then restricted using 50U Xbal for 2 hours at 37° C. The reaction wasstopped by the removal of reaction buffer and the addition of0.5×Tris-borate EDTA buffer (TBE). The DNA samples were electrophoresedin 1% SeaKem gold agarose in 0.5×TBE buffer with a contour-clampedhomogeneous electric field device (CHEF MAPPER, Bio-Rad, Hercules,Calif.). After electrophoresis for 18 hours at 6.0 V/cm with pulse timesof 2.16 to 54.17 seconds, linear ramping and an electric field angle of120 at 14° C., the gels were stained with ethidium bromide. The bandswere visualized and photographed with UV transillumination.

Antibiotic resistance profiles of all unique isolates were obtainedusing Sensititre gram-positive and gram-negative MIC plates (TREKDiagnostic Systems, Inc. Westlake, Ohio).

Results

A total of 1097 bacterial colonies were isolated from the feces ofcattle determined not to excrete Salmonella spp. These bacteria wereinitially screened for their ability to inhibit the growth of, or kill athree-strain mixture of S. Typhimurium DT104 in vitro, and 45 weredetermined to be inhibitory (Table 2), one through the paper disk assay(FIGS. 1A and 1B.), and 44 via the agar-spot test 9 (FIGS. 2A and 2B.).The size and clarity of the zones of inhibition varied with the type ofmedia and the competitive inhibition candidate. TABLE 2 Initialscreening of potential competitive inhibition bacteria with inhibitoryactivity against 3 strains of S. Typhimurium DT104^(a). Overlay IsolateDate of Disk Assay^(b) Assay^(c) No. Source Isolation XLD TSA MAC TSA1-1 Dairy cow Sep. 10, 2001 − − + − 3-7 Dairy cow Sep. 10, 2001 − − − +4-4 Dairy cow Sep. 10, 2001 − + + − 4-5 Dairy cow Sep. 10, 2001 − − + +5-3 Dairy cow Sep. 10, 2001 − − + − 6-8 Dairy cow Sep. 10, 2001 − − − +7-7 Dairy cow Sep. 10, 2001 − − + − 8-7 Dairy cow Sep. 10, 2001 − − + −9-2 Dairy cow Sep. 10, 2001 − − + − 11-1  Dairy cow Sep. 10, 2001 − − +− 12-5  Dairy cow Sep. 10, 2001 − − + − 13-2  Dairy cow Sep. 10, 2001 −− + − 13-6  Dairy cow Sep. 10, 2001 − − + − 15-3  Dairy cow Sep. 10,2001 − − + − 15-6  Dairy cow Sep. 10, 2001 − − + − 16-2  Dairy cow Sep.10, 2001 − − + − 16-6  Dairy cow Sep. 10, 2001 − − + − 16-10 Dairy cowSep. 10, 2001 − − + − 18-4  Dairy cow Sep. 10, 2001 − − + − 18-6  Dairycow Sep. 10, 2001 − − + − 21-6  Dairy cow Sep. 10, 2001 − − + − 21-9 Dairy cow Sep. 10, 2001 − − + − 23-5  Dairy cow Sep. 10, 2001 − − + −24-2  Dairy cow Sep. 10, 2001 − − + − 25-10 Beef calf Oct. 16, 2001 −− + + 29-5  Beef calf Oct. 16, 2001 − − + + 30-1  Beef calf Oct. 16,2001 − − + + 30-5  Beef calf Oct. 16, 2001 − − + + 31-6  Beef cow Oct.16, 2001 ++ ++ − − 35-3  Beef cow Oct. 16, 2001 − − + − 39-3  Beef cowOct. 16, 2001 − − + − 44-2  Beef cow Oct. 16, 2001 − − + − 44-4  Beefcow Oct. 16, 2001 − − + − 47-10 Beef cow Oct. 16, 2001 − − − ++ 50-10Beef cow Oct. 16, 2001 − − − ++ 51-2  Beef cow Oct. 16, 2001 − − + +58-7  Beef cow Oct. 16, 2001 − − + + 58-9  Beef cow Oct. 16, 2001 − − −++ 59-9  Beef cow Oct. 16, 2001 − − − ++ small 59-9  Beef cow Oct. 16,2001 − − − ++ big 66-3  Beef cow Jan. 15, 2002 − − + − 71-8  Beef cowJan. 15, 2002 − − + +++ 76-9  Beef cow Jan. 15, 2002 − − ++ 101-1  Beefcow Jan. 29, 2002 − − + + 106-2  Beef cow Jan. 29, 2002 − − + +^(a)Strains include: 8748A-1, 11942A-1, 62^(b)Zone of inhibition: ++ = >2.0 mm, + = <2.0 mm, −= no zone^(c)Zone of inhibition: +++ = >10 mm, ++ = >5.0 mm, + = <5.0 mm, −= nozone

The 45 candidates were screened for in vitro inhibitory activity againstan additional nine isolates of S. Typhimurium DT104, and 30 weredetermined to be inhibitory (Table 3 and Table 4), one through the paperdisk assay, and 29 through the agar-spot assay. These 30 candidates werethen screened for antimicrobial activity against the isolated Salmonellaspp. isolates from the bovine feces. Only six gram-positive bacteriaproduced any degree of inhibitory activity against all 10 strains asdemonstrated by the agar spot test. Three gram-negative isolates wereinhibitory to five of the ten isolated strains (Table 5 and Table 6).TABLE 3 Screening of potential competitive inhibition bacteria withinhibitory activity against 5 strains of S. Typhimurium DT104^(a).Isolate Disk Assay^(b) Overlay Assay^(c) No. XLD TSA MAC TSA  1-1 − − +−  3-7 − − + −  4-4 − − + −  4-5 − − + −  5-3 − − + +  6-8 − − + −  7-7− − + −  8-7 − − − −  9-2 − − + −  11-1 − − + −  12-5 − − + −  13-2 −− + −  13-6 − − + −  15-3 − − + −  15-6 − − + −  16-2 − − + −  16-6 −− + −  16-10 − − − −  18-4 − − + −  18-6 − − + −  21-6 − − + −  21-9 − −− +  23-5 − − + −  24-2 − − + +  25-10 − − + −  29-5 − − + −  30-1 − − +−  30-5 − − + +  31-6 ++ ++ + −  35-3 − − + −  39-3 − − − −  44-2 − − −−  44-4 − − − −  47-10 − − − +++  50-10 − − − −  51-2 − − + −  58-7 −− + −  58-9 − − − +++  59-9 small − − − +++  59-9 big − − − +++  66-3 −− + −  71-8 − − − +++  76-9 − − − +++ 101-1 − − + − 106-2 − − + −^(a)Strains include: AI45025, MH2538, 99-103712-5, NE14055, 12-410^(b)Zone of inhibition: ++ = >2.0 mm, + = <2.0 mm, − = no zone^(c)Zone of inhibition: +++ = >10 mm, ++ = >5.0 mm, + = <5.0 mm, − = nozone

TABLE 4 Screening of potential competitive inhibition bacteria withinhibitory activity against 4 strains of S. Typhimurium DT104^(a).Isolate Disk Assay^(b) Overlay Assay^(c) No. XLD TSA MAC TSA  1-1 − − +−  3-7 − − + +  4-4 − − + −  4-5 − − + −  5-3 − − − +  6-8 − − + −  7-7− − + −  8-7 − − − −  9-2 − − + −  11-1 − − + −  12-5 − − + −  13-2 −− + −  13-6 − − + −  15-3 − − − +  15-6 − − + −  16-2 − − + −  16-6 −− + +  16-10 − − − −  18-4 − − + −  18-6 − − − −  21-6 − − − −  21-9 − −− +  23-5 − − − −  24-2 − − − −  25-10 − − − −  29-5 − − − −  30-1 − − +−  30-5 − − + −  31-6 ++ ++ + −  35-3 − − + −  39-3 − − + −  44-2 − − +−  44-4 − − − −  47-10 − − − +++  50-10 − − − −  51-2 − − + −  58-7 −− + −  58-9 − − − +++  59-9 small − − − +++  59-9 big − − − +++  66-3 −− + −  71-8 − − − +++  76-9 − − − +++ 101-1 − − + + 106-2 − − + −^(a)Strains include: 12993-k, 2748-k, 520-k, 4698-k^(b)Zone of inhibition: ++ = >2.0 mm, + = <2.0 mm, − = no zone^(c)Zone of inhibition: +++ = >10 mm, ++ = >5.0 mm, + = <5.0 mm, − = nozone

TABLE 5 Screening of potential competitive inhibition bacteria withinhibitory activity against 5 strains of Salmonella spp.^(a) isolatedfrom beef cattle in Georgia. Isolate Disk Assay^(b) Overlay Assay^(c)No. XLD TSA MAC TSA  1-1 − − − −  3-7 − − − −  4-4 − − − −  4-5 − − − − 5-3 − − − −  6-8 − − − −  7-7 − − − −  8-7 − − − −  9-2 − − − −  11-1 −− − −  12-5 − − + −  13-2 − − − −  13-6 − − − −  15-3 − − − −  15-6 − −− −  16-2 − − − −  16-6 − − − −  16-10 − − − −  18-4 − − − −  18-6 − − −−  21-6 − − − −  21-9 − − − −  23-5 − − − −  24-2 − − − −  25-10 − − − − 29-5 − − − −  30-1 − − − −  30-5 − − − −  31-6 ++ ++ + −  35-3 − − − − 39-3 − − − −  44-2 − − − −  44-4 − − − −  47-10 − − − +++  50-10 − − −−  51-2 − − − −  58-7 − − − −  58-9 − − − +++  59-9 small − − − +++ 59-9 big − − − +++  66-3 − − − −  71-8 − − − +++  76-9 − − − +++ 101-1− − + − 106-2 − − − −^(a)Strains include: S. Newport 55, S. Newport 57, S. Bareilly 73, S.Mbandaka 74, S. Newport 88^(b)Zone of inhibition: ++ = >2.0 mm, + = <2.0 mm, − = no zone^(c)Zone of inhibition: +++ = >10 mm, ++ = >5.0 mm, + = <5.0 mm, − = nozone

TABLE 6 Screening of potential competitive inhibition bacteria withinhibitory activity against 5 strains of Salmonella spp.^(a) isolatedfrom beef cattle in Georgia. Isolate Disk Assay^(b) Overlay Assay^(c)No. XLD TSA MAC TSA  1-1 − − − −  3-7 − − − −  4-4 − − − −  4-5 − − − − 5-3 − − − −  6-8 − − − −  7-7 − − − −  8-7 − − − −  9-2 − − − −  11-1 −− − −  12-5 − − − −  13-2 − − − −  13-6 − − − −  15-3 − − − −  15-6 − −− −  16-2 − − − −  16-6 − − − −  16-10 − − − −  18-4 − − − −  18-6 − − −−  21-6 − − − −  21-9 − − − −  23-5 − − − −  24-2 − − − −  25-10 − − − − 29-5 − − − −  30-1 − − − −  30-5 − − − −  31-6 − − − −  35-3 − − − − 39-3 − − − −  44-2 − − − −  44-4 − − − −  47-10 − − − +++  50-10 − − −−  51-2 − − − −  58-7 − − − −  58-9 − − − +++  59-9 small − − − +++ 59-9 big − − − +++  66-3 − − − −  71-8 − − − +++  76-9 − − − +++ 101-1− − − − 106-2 − − − −^(a)Strains include: S. Newport 88, S. Montevideo 90, S. Meleagridis 92,and monophasic Salmonella spp. 102 and 103.^(b)Zone of inhibition: ++ = >2.0 mm, + = <2.0 mm, − = no zone^(c)Zone of inhibition: +++ = >10 mm, ++ = >5.0 mm, + = <5.0 mm, − = nozone

The initial pH of TSB was 7.38, the pH of the medium decreased from 0.82to 1.70 pH units occurred with the growth of each of the 30 competitiveinhibition isolates that were active against all 12 strains of S.Typhimurium DT104. A decrease in pH also occurred with the growth of 12S. Typhimurium DT104 strains and ranged from 1.10 to 1.37 pH units. ThepH on MAC plates either increased slightly or decreased slightly nearthe competitive inhibition colonies, with a maximum pH increase of 0.22pH units, and a maximum pH decrease of 0.54 pH units. The pH on TSAplates also increased slightly with the growth of some competitiveinhibition candidates and decreased slightly with others, with a maximumpH increase of 0.34 pH units, and a maximum pH decrease of 0.08 pHunits.

Generation times of S. Typhimurium DT104 in TSB averaged 25 minutes,those of gram-negative competitive inhibition isolates ranged from 25minutes to 50 minutes. The generation times of gram-positive competitiveinhibition isolates ranged from 38 minutes to 52 minutes.

The six gram-positive bacteria isolates were catalase-positive andoxidase-negative (Table 7). The API 50CH gave only doubtful profileswith the CHL media. With the CHB/E media gave very good identificationof isolate 71-8 as Bacillus circulans, good identification of isolates58-9 and 76-9 as Bacillus circulans, acceptable identification of 47-10as Bacillus circulans, low discrimination for isolate 59-9 small asBacillus circulans, and a doubtful profile for 59-9 big as Bacilluscirculans. Each isolate was confirmed to be a spore producer. Antibioticresistance profiles varied, with all strains being resistant tocefoxitin and ceftiofur, some strains being resistant to streptomycin,varying strains having intermediate resistance and resistance toceftriaxone, all strains having intermediate resistance to tetracyclineand some strains having intermediate resistance to chloroamphenicol.TABLE 7 Selected characteristics of potential gram-positive competitiveinhibition bacteria with inhibitory activity against 12 strains of S.Typhimurium DT104, and 10 strains of Salmonella spp. isolated fromcattle. Isolate No. Identification Antibiotic resistance^(ab) 47-10Bacillus circulans CeCfCtS 58-9 Bacillus circulans CeCfCS 59-9 smallBacillus circulans* CeCfS 59-9 big Bacillus circulans* CeCfS 71-8Bacillus circulans CeCfCtS 76-9 Bacillus circulans CeCfS^(a)Ce = cefoxitin, Cf = ceftiofur, Ct = cefiriaxone, S = streptomycin,C = chloroamphenicol^(b)Screened against, amikacin, amoxicillin/clavulanic acid, ampicillin,apramycin, cefoxitin, ceftiofur, ceftriazone, cephalothin,chloramphenicol, ciprofloxacin, gentamicin, inipenem, kanamycin,nalidixic acid, streptomycin, sulphamethoxazole,# tetracycline, trimethoprim/sulphamethoxazole*Doubtful profiles/low discrimination by API 50 CH screening

The 24 gram-negative competitive inhibition (CI) bacteria wereidentified through biochemical testing using the API 20E Assay (Table8). Twenty-two E. coli, one Serratia fonticola, and one Enterobactercloacae were identified. Genomic DNA subtyping revealed 17 differentprofiles among the 22 E. coli isolates. FIG. 3 shows the resultingelectrophoretic pattern of the DNA samples wherein lanes 1 and 8 are E.coli O157:H7, G5244, lane 2 is isolate 5-3, lane 3 is isolate 12-5, lane4 is isolate 13-6, lane 5 is isolate 13-2, lane 6 is isolate 30-5, lane7 is isolate 30-1, lane 9 is isolate 15-6, lane 10 is isolate 16-2 andlane 11 is isolate 18-4. Antibiotic resistance profiling revealed thatall the strains had some level of resistance to tetracycline. Otherresistance to antibiotics varied among strains. TABLE 8 Selectedcharacteristics of potential gram-negative competitive inhibitionbacteria with inhibitory activity against 12 strains of S. TyphimuriumDT104 and isolated from cattle. PFGE DNA Antibiotic Isolate No.Identification subtype^(a) resistance^(bc)  1-1 E. coli Unique None  3-7S. fonticola Unique None  4-5 E. cloacae Unique Am, A, Ce, Cp  5-3 E.coli Unique None  6-8 E. coli Unique None  7-7 E. coli Unique None  9-2E. coli Unique None  11-1 E. coli Unique None  12-5 E. coli Same as 5-3None  13-2 E. coli Unique SSuT  13-6 E. coli Same as 13-2 SsuT  15-6 E.coli Unique None  16-2 E. coli Same as 15-6 None  16-6 E. coli UniqueNone  18-4 E. coli Same as 15-6 None  30-1 E. coli Unique Intermediate C 30-5 E. coli Same as 30-1 Intermediate C  31-6 E. coli Unique T  44-2E. coli Unique Intermediate C  51-2 E. coli Unique None  58-7 E. coliUnique None  66-3 E. coli Unique Su 101-1 E. coli Unique A, Cp 106-2 E.coli Unique None^(a)DNA subtyping as determined by PEGE; unique indicates that the PFGEpulsotype is different from those of the other strains in this study^(b)Am = amoxicillin/clavulancic acid, A = ampicillin, C =chloramphenicol, Ce = cefoxitin, Cp = cephalothin, S = streptomycin, Su= sulphamethoxazole, T = tetracycline^(c)Screened against, amikacin, amoxicillin/clavulanic acid, ampicillin,apramycin, cefoxitin, ceftiofur, ceftriazone, cephalothin,chloramphenicol, ciprofloxacin, gentamicin, inipenem, kanamycin,nalidixic acid, streptomycin, sulphamethoxazole,# tetracycline, trimethoprim/sulphamethoxazole

EXAMPLE 3 Competitive Growth in Feces

Methods

Preparation of competitive inhibition bacteria for inoculation intofeces. To facilitate enumeration of the competitive inhibition bacteria,all gram-negative bacterial isolates were selected for resistance tonalidixic acid (50 μg/ml) by exposure to serially (1:2) increasedconcentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25 and 50μg/ml) of nalidixic acid in TSB every 24 hours at 37° C. A single colonyof each strain of nalidixic acid-resistant, gram-negative, competitiveinhibition bacteria was transferred to 10 ml of TSB containing nalidixicacid (50 μg/ml) and incubated for 24 hours at 37° C. A 0.1 ml portionwas transferred to 10 ml of TSB and incubated for 16 hours at 37° C.Bacteria were then sedimented by centrifugation (4000×g, 10 min), washedthree times in 0.1% phosphate buffered saline, pH 7.2 (PBS) and thenresuspended in PBS to an optical density of 0.5 at 640 nm (ca. 10⁸CFU/ml). Nineteen gram-negative isolates were combined at equalpopulations. Two levels of inocula (10⁵ and 10⁸ CFU of gram-negativecompetitive inhibition isolates strains per g of feces) were used.

Gram-positive competitive inhibition isolates were transferred to 10 mlof TSB and incubated for 16 hours at 37° C. Bacteria were thensedemented by centrifugation (4000×g, 10 min), washed three times in0.1% phosphate buffered saline, pH 7.2 (PBS) and then resuspended in PBSto an optical density of 0.5 at 640 nm (ca. 10⁸ CFU/ml). Sixgram-positive strains were combined at equal populations. Two levels ofinocula (10⁵ and 10⁸ CFU of gram-positive competitive inhibitionisolates per g of feces) were used.

A four-strain mixture of S. Typhimurium DT104, including strains 4698-K,11942A1, 8748A1 and 62, which were previously described, was used. Eachstrain was grown in 10 ml of TSB held for 16 hours at 37° C. Bacteriawere then sedimented by centrifugation (4000×g, 10 min), washed threetimes in 0.1% phosphate buffered saline, pH 7.2 (PBS) and thenresuspended in PBS to an optical density of 0.5 at 640 nm (ca. 10⁸CFU/ml). The four S. Typhimurium DT104 strains were combined at equalpopulations. Two levels of inocula (10³ and 10⁵ CFU of S. TyphimuriumDT104 per g of feces) were used.

A four strain mixture of Salmonella enteritidis serovar Newport,including, strains S55, S57, S78, and S88, which were all characterizedin this study, was used. The four-strain cell suspension was preparedaccording to the same procedures described above for S. TyphimuriumDT104. Two levels of inocula (10⁵ and 10⁸ CFU of S. Newport per g offeces) were used.

Feces. Ten healthy beef cattle over the age of one year were used as thesources of feces. Fecal samples, which were obtained in June, werecollected into 50 ml Falcon tubes, and transported to the laboratory at5° C. All samples were screened for Salmonella spp. by the proceduredescribed above. All feces were mixed well in stomacher bags at mediumspeed for 5 minutes.

Inoculation of the feces with S. Typhimurium DT104, S. Newport andcompetitive inhibition bacteria. The inocula of S. Typhimurium DT104 orS. Newport, and gram-negative competitive inhibition bacteria orgram-positive competitive inhibition bacteria (total 2 ml) mixtures atthe appropriate dilution were added to 18 g of feces in sterilestomacher bags and mixed in a stomacher at medium speed for five minutesto obtain the desired bacterial concentrations.

Fecal sample inoculations included, 10⁵ S. Typhimurium DT104/g and 10⁸gram-negative competitive inhibition bacteria/g, 10³ S. TyphimuriumDT104/g and 10⁵ gram-negative competitive inhibition bacteria/g, 105 S.Typhimurium DT104/g and 10⁸ gram-positive competitive inhibitionbacteria/g, 10³ S. Typhimurium DT104/g and 10⁵ gram-positive competitiveinhibition bacteria/g, 105 S. Newport/g and 10⁸ gram-positivecompetitive inhibition bacteria/g, and 10³ S. Newport/g and 10⁵gram-positive competitive inhibition bacteria/g. Controls included bothinoculation levels of gram-negative competitive inhibition bacteria,gram-positive competitive inhibition bacteria, S. Typhimurium DT104, S.Newport and total aerobic counts.

Incubation and Sampling. Inoculated fecal samples were held underaerobic conditions at 21° and 37° C. Duplicate samples were obtained at0, 1, 3, 5, 7, 14 and 21 days post-inoculation. Fecal samples (1 g) wereserially diluted (1:10) in 0.1% peptone and assayed for S. TyphimuriumDT104 or S. Newport counts by direct plating 0.1 ml portions onto XLDcontaining ampicillin (32 μg/ml), tetracycline (16 μg/ml) andstreptomycin (64 μg/ml) (XLD+). Plates were incubated for 24 hours at37° C. When Salmonella was not detectable by direct plating, 1 g samplesof feces mixed with 0.1% peptone were added to 10 ml of double strengthlactose broth for enrichment cultures at 35° C. for 24 hours. Enrichmentcultures were subsequently plated onto XLD+ and incubated at 37° C. for24 hours. Gram-negative competitive inhibition bacteria were enumeratedby direct plating 0.1 ml portions onto MAC containing nalidixic acid (50μg/ml). Plates were incubated for 24 hours at 37° C. Gram-positivecompetitive inhibition bacteria were enumerated by direct plating 0.1 mlportions onto TSA, incubating for 24 hours at 37° C., and subtractingtotal aerobic counts obtained for that day of the study. pH values weredetermined for 1 g fecal samples mixed with 9 ml of 0.1% peptone. Alltests were performed in duplicate and the entire study was performed intriplicate.

Statistical analysis. The Statistical Analysis System (SAS) computerstatistical package (SAS Institute, Cary, N.C.) was used for analysis ofdata with Duncan's multiple range tests to determine if significantdifferences (P<0.05) in populations of S. Typhimurium DT104 existbetween mean population values.

Results

The average initial aerobic plate count of the fecal samples was 2.6×10⁹CFU/g, and the average initial pH was 7.1. No Salmonella serovars weredetected in the feces before inoculation. At 37° C., all the Salmonellapopulations increased about 2 log₁₀ CFU/g one-day post inoculation(FIGS. 4A-4C, wherein open diamonds represent DT104, closed trianglerepresent S. Newport, low inoculum and open triangles represent S.Newport, high inoculum). No significant differences were observed witheither of the inoculations of competitive inhibition bacteria against S.Typhimurium DT104 at both inoculation levels during the 21-day period(FIGS. 4A and 4B, wherein open squares represent DT104 withgram-negative CI bacteria and closed circles represent DT104 withgram-positive CI bacteria). At 37° C., a significant difference (P>0.05)was observed with the low-level inoculations of gram-positivecompetitive inhibition isolates with S. Newport at days 3 and 5, and atthe high level inoculation at day 21 (FIG. 4C, wherein x represents S.Newport with gram-positive low inoculum and ζ represents S. Newport withgram-positive high inoculum). The pH of the feces increased slightly(ca. pH of Salmonella only−7.25, pH Salmonella and CI bacteria=7.58) forall samples during the incubation period.

At 21° C., a population increase of 1 to 4 log₁₀ Salmonella/g was seenfollowing the first day of growth (FIGS. 5A-5C, wherein open diamondsrepresent DT104, closed triangle represent S. Newport, low inoculum andopen triangles represent S. Newport, high inoculum). The low-levelinoculation of gram-negative competitive inhibition bacteria did notsignificantly reduce (P>0.05) S. Typhimurium DT104 growth when comparedto the control (FIG. 5A, wherein open squares represent DT104 withgram-negative CI bacteria and closed circles represent DT104 withgram-positive CI bacteria). The high inoculation level of gram-negativecompetitive inhibition bacteria significantly reduced S. TyphimuriumDT104 populations at day 5 only (FIG. 5B, wherein open squares representDT104 with gram-negative CI bacteria and closed circles represent DT104with gram-positive CI bacteria). A significant reduction (P>0.05) of S.Typhimurium DT1 04 occurred at day five of the low inoculation level ofgram-positive competitive inhibition bacteria. No significant reductionsoccurred at this temperature with the high inoculation level ofgram-positive competitive inhibition bacteria. The gram-positivecompetitive inhibition bacteria did not significantly reduce (P>0.05)the growth/survival of S. Newport at 21° C. (FIG. 5C, wherein xrepresents S. Newport with gram-positive low inoculum and ζ representsS. Newport with gram-positive high inoculum). The pH increase slightlyfor all Salmonella control samples (ca. pH=7.49), and decreased slightlyfor Salmonella and CI bacteria samples (ca. pH=7.02 ) during theincubation period.

While the disclosure is susceptible to various modifications andalternative forms, specific exemplary embodiments thereof have beenshown by way of example in the drawings and has herein be described indetail. It should be understood, however, that there is no intent tolimit the disclosure to the particular forms disclosed, but on thecontrary, the intention is to cover all modifications, equivalents, andalternatives falling within the spirit and scope of the disclosure.

There are a plurality of advantages of the present disclosure arisingfrom the various features of the apparatus and methods described herein.It will be noted that alternative embodiments of the apparatus andmethods of the present disclosure may not include all of the featuresdescribed yet still benefit from at least some of the advantages of suchfeatures. Those of ordinary skill in the art may readily devise theirown implementations of an apparatus and method that incorporate one ormore of the features of the present disclosure and fall within thespirit and scope of the present disclosure.

1. A method for preventing or treating the carriage of Salmonellastrains that cause human salmonellosis in a food production animalcomprising administering an effective amount of one or more strains ofprobiotic bacteria, wherein the probiotic bacteria inhibits the growthof the Salmonella strains that cause human salmonellosis.
 2. The methodof claim 1 wherein the Salmonella strain is the Salmonella entericaserovar Typhimurium DT104 or Newport.
 3. The method of claim 1 whereinthe probiotic bacteria strain is selected from the group consisting ofisolate 31-6, 47-10, 50-10, 58-9, 59-9 small, 59-9 big, 71-8 and 76-9.4. The method of claim 1 wherein the probiotic bacteria strain isselected from the group consisting of isolate 31-6, 76-9 and 58-9. 5.The method of claim 1 wherein the food production animal is a ruminantanimal.
 6. The method of claim 5 wherein the ruminant animal is a beefcow or a dairy cow.
 7. The method of claim 1 wherein the food productionanimal is a chicken or turkey.
 8. The method of claim 1 wherein theprobiotic bacteria is administered to the animal by applying thebacteria to an animal's feed or water.
 9. The method of claim 1 whereinthe probiotic bacteria is administered to the animal as an innoculantpaste.
 10. A composition for preventing or treating the carriage ofSalmonella strains that cause human salmonellosis in a food productionanimal comprising probiotic bacteria that inhibits the growth of theSalmonella strains, a carrier and an animal's feed or water.
 11. Thecomposition of claim 10 wherein the probiotic bacteria is selected fromthe group consisting of isolates 31-6, 47-10, 50-10, 58-9, 59-9 small,59-9 big, 71-8 and 76-9.
 12. The composition of claim 10 wherein theprobiotic bacteria is selected from the group consisting of isolates31-6, 76-9, and 58-9.
 13. The composition of claim 10 wherein the feedis liquid or solid feed.